Use of an interleukin 1 receptor antagonist and/or pyrrolidinedithiocarbamate for the treatment or prophylaxis of type 2 diabetes

ABSTRACT

Substances that inhibit the action of the members of the IL-1β/NF-κB pathway can be used for protecting and preserving β-cell mass and function in prediabetic and diabetic type 2 patients. Specifically, the present invention relates to the use of an Interleukin 1 receptor antagonist (IL-1Ra) and/or pyrrolidinedithiocarbamate (PDTC) for the treatment or prophylaxis of type 2 diabetes, as well as a method for the treatment of type 2 diabetes.

FIELD OF THE INVENTION

The present invention relates to the use of an Interleukin 1 receptorantagonist (IL-1Ra) and/or pyrrolidinedithiocarbamate (PDTC) for thetreatment or prophylaxis of type 2 diabetes, as well as a method for thetreatment of type 2 diabetes.

BACKGROUND OF THE INVENTION

Type 2 diabetes mellitus results from an inadequate adaptation of thefunctional pancreatic β-cell mass in the face of insulin resistance. Inturn, hyperglycemia per se has secondary adverse effects on β-cells.Indeed, several studies have shown that chronic elevation of bloodglucose concentration impairs β-cell function, leading to the concept of“glucotoxicity” (1-7). Moreover, elevated glucose concentrations induceβ-cell apoptosis in cultured islets from diabetes-prone Psammomys obesus(8), from human islets (9;10) and at higher concentrations in rodentislets (8;11;12). Various molecular mechanisms have been proposed tounderlie glucose-induced β-cell dysfunction, including formation ofadvanced glycation end products (13), direct impairment of insulin genetranscription and proinsulin biosynthesis (14;15) and reduced bindingactivity of pancreatic duodenal homeobox 1 (PDX-1) (7). Recently, thepresent inventor and co-workers proposed a mechanism underlyingglucose-induced B-cell apoptosis in human islets, which involvesup-regulation of Fas receptors by elevated glucose levels (9). However,the mediator of glucose-induced Fas-expression and its role inglucotoxicity remains unknown.

Interleukin 1 receptor antagonist (IL-1Ra) is a mature glycoprotein of152 amino acid (aa) residues. The protein has a native molecular weightof 25 kDa, and the molecule shows limited aa sequence homology to IL-1α(19%) and IL-1β (26%).

The effects produced by IL-1α and IL-1β result from the binding of thesefactors to two distinct cell surface receptors, IL-1R types I and II.Recent results suggest that only the type I receptor is capable oftransducing a signal and producing a biological effect. The inhibitoryaction of IL-1Ra results from its binding to the type I IL-1R. Althoughthis binding is of high-affinity (Kd=200 pM), it does not result inreceptor activation (signal transduction), an effect attributed to thepresence of only one receptor binding motif on IL-1Ra vs. two suchmotifs on IL-1α and IL-1β. Since the affinity of IL-1Ra for the type Ireceptor is comparable to that for IL-1α and IL-1β, down-regulation ofIL-1 activity seems to be due to competitive inhibition. Notably, IL-1Raalso binds to the non-signal transducing type II IL-1R (Kd=7 nM), butwith considerably lower affinity than that shown by IL-1β (Kd=0.3−2.0nM). This makes sense teleologically in that two mechanisms designed toinhibit the actions of IL-1β (i.e., IL-1Ra binding to IL-1R type I andIL-1β binding to IL-1R type II) should not compete with each other.

It has been proposed to use IL-1β for mediating both impaired functionand destruction of pancreatic β-cell during the development ofautoimmune type 1 diabetes (16). In keeping with this, treatment ofrodent islets with IL-1β results in a potent inhibition of insulinsecretion followed by islet destruction (17-23). In human islets, IL-1βhas further been shown to impair insulin release and to induce Fasexpression enabling Fas-triggered apoptosis (9;24-28). Finally,activation of the nuclear transcription factor NF-κB is required forIL-1β-induced Fas expression (29-31). Part of these IL-1β effects arereminiscent of the toxic effects of elevated glucose concentrations.

Zaitsev et al. (60) propose the treatment of type 2 diabetes withimidazoline compounds.

SUMMARY OF THE INVENTION

The inventor and co-workers believe that glucose may induce IL-1βsecretion from β-cells in the absence of an autoimmune process. Thepresent inventor and co-workers has identified β-cells as the cellularsource of glucose-induced IL-1β in cultured human islets, and hasconfirmed this using tissue sections from the pancreas of type 2diabetic patients and of Psammomys obesus. The role of such endogenouslyproduced IL-1β in β-cell glucotoxicity has also been explored.

In type 2 diabetes, chronic hyperglycemia is suggested to be detrimentalto pancreatic β-cells, causing impaired insulin secretion.Interleukin-1β (IL-1β) is a pro-inflammatory cytokine acting during theautoimmune process of type 1 diabetes. IL-1β inhibits β-cell functionand promotes Fas-triggered apoptosis in part by activating thetranscription factor NF-κB. Recently, the present inventor andco-workers have shown that increased glucose concentrations also induceFas-expression and β-cell apoptosis in human islets. The aim of thepresent study was to test the hypothesis that IL-1β may mediate thedeleterious effects of high glucose on human β-cells. In vitro exposureof islets from nondiabetic organ donors to high glucose levels resultedin increased production and release of IL-1β, followed by NF-κBactivation, Fas up-regulation, DNA-fragmentation and impaired β-cellfunction. The interleukin-1 receptor antagonist IL-1Ra protectedcultured human islets from these deleterious effects. β-cells themselveswere identified as the islet cellular source of glucose-induced IL-1β.In vivo, IL-1β producing β-cells were observed in pancreatic sections oftype 2 diabetic patients but not in nondiabetic control subjects.Similarly, IL-1β was induced in β-cells of the gerbil Psammomys obesusduring development of diabetes. Treatment of the animals with phlorizinnormalized plasma glucose and prevented β-cell expression of IL-1β.These findings implicate an inflammatory process in the pathogenesis ofglucotoxicity in type 2 diabetes and identify the IL-1β/NF-κB pathway asa target to preserve β-cell mass and function in this condition.

Thus, the present inventor has found that Interleukin 1 receptorantagonist (IL-1Ra) and/or the NF-κB inhibitorpyrrolidinedithiocarbamate (PDTC) can be useful for the treatment orprophylaxis of type 2 diabetes.

One aspect of the invention relates to the use of an Interleukin 1receptor antagonist (IL-1Ra) for the preparation of a medicament for thetreatment or prophylaxis of type 2 diabetes in a mammal.

Another aspect of the invention relates to the use ofpyrrolidinedithiocarbamate (PDTC) for the preparation of a medicamentfor the treatment or prophylaxis of type 2 diabetes in a mammal.

A third aspect of the invention relates to the combined use of anInterleukin 1 receptor antagonist (IL-1Ra) andpyrrolidinedithiocarbamate (PDTC) for the preparation of a medicamentfor the treatment or prophylaxis of type 2 diabetes in a mammal.

A fourth aspect of the invention relates to the method of treating orprophylactically suppressing type 2 diabetes, wherein the methodcomprises administering to a mammal in need thereof a sufficient amountof an Interleukin 1 receptor antagonist (IL-1Ra).

A fifth aspect of the invention relates to the method of treating orprophylactically suppressing type 2 diabetes, wherein the methodcomprises administering to a mammal in need thereof a sufficient amountof pyrrolidinedithiocarbamate (PDTC).

A sixth aspect of the invention relates to the method of treating orprophylactically suppressing type 2 diabetes, wherein the methodcomprises administering to a mammal in need thereof a sufficient amountof a combination of an Interleukin 1 receptor antagonist (IL-1Ra) andpyrrolidinedithiocarbamate (PDTC).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1—Glucose induces IL-1β expression and release in human islets.

A, Secretion of IL-1β from human islets cultured on extracellularmatrix-coated dishes for 4 days in 5.5, 11.1 or 33.3 mM D-glucose or in5.5 mM D-glucose plus 27.8 mM L-glucose. Each bar represents the mean ofeight experiments±SE from eight separate donors. *, P<0.01 relative toislets at 5.5 mM glucose. B, Secretion of IL-1β from human islets during44-hour cultured in a suspension at 5.5 or 33.3 mM D-glucose. Data werecollected from four tubes per treatment in two separate experiments fromtwo donors. Data are represented as mean±SE. *, P<0.01 relative toislets at 5.5 mM glucose. C, Immunoblotting of pro-IL-1β, IL-1β andactin. Human islets cultured in a suspension at 5.5 or 33.3 mM glucosewere analysed after 44 hours of incubation. One experiment out of elevenfrom eleven donors is shown. In seven experiments, glucose inducedIL-1β, in three experiments, IL-1β remained unchanged and in one, it wasdecreased. D, RT-PCR detection and quantification of IL-1β mRNAexpression. Total RNA was isolated from human islets cultured for 44hours in a medium containing 5.5 or 33.3 mM glucose. In the Light Cyclerquantitative PCR system, the level of IL-1β expression was normalizedagainst GAPDH and the results expressed as mRNA levels relative tocontrol incubations at 5.5 mM. Results are shown ±SE for 6 independentexperiments from 6 donors. *, P<0.05 relative to islets at 5.5 mMglucose.

FIG. 2—Expression of IL-1β by human β-cells exposed to a diabeticmilieu.

Double immunostaining for IL-1β (B, D) and insulin (A, C) in humanislets cultured on extracellular matrix-coated dishes and exposed for 4days to media containing 5.5 mM glucose (A, B) or 33.3 mM glucose (C,D). Double immunostaining for IL-1β (F, H) and insulin (E, G) in tissuesections of pancreases from a non-diabetic patient (E, F) and from apatient with type 2 diabetes (G, H). In situ hybridization for IL-1βmRNA (J, L, N) double immunostained for insulin (I, K, M) in tissuesections of pancreases from a patient with type 2 diabetes withanti-sense probe (L) and with sense probe (negative control) (N), andfrom a non-diabetic patient using anti-sense probe (J). Immunostainingfor IL-1β in lipopolysaccharide (LPS) treated macrophages (positivecontrol) (O). 250-fold magnification.

FIG. 3—β-cell expression of IL-1β during development of diabetes inPsammomys obesus.

Double immunostaining for IL-1β (B, D, F) and insulin (A, C, E) intissue sections of pancreases from a fasted Psammomys obesus onlow-energy diet (blood glucose 4 mM) (A, B), from an animal on ahigh-energy diet for 8 days without (blood glucose 13.6 mM) (C, D) andwith injections of the glucopenic drug phlorizin (blood glucose 5.6 mM)(E, F). 250-fold magnification.

FIG. 4—Glucose decreases β-cell's IκB expression and inducesIL-1β-meditated NF-κB activation, Fas expression and DNA fragmentation.

A, Relative NF-κB activity. Human islets were cultured in a suspensionfor 44 hours in 5.5 or 33.3 mM glucose alone or in the presence ofIL-1β, IL-1Ra or both. HeLa cells, stimulated with 5 ng/ml IL-1α, wereused as positive control. Each bar represents the mean of threeexperiments±SE from three separate donors. *, P<0.05 relative to isletsat 5.5 mM glucose. B, Immunoblotting of NF-κB (p65), Fas and actin.Human islets cultured in a suspension at 5.5 or 33.3 mM glucose with andwithout IL-1β or IL-1Ra were analyzed after 44 hours of incubation. Theantibodies were blotted on the same membrane after stripping. One out ofthree experiments from three donors is shown. Each experiment displayedsimilar results. C, Double immunostaining for IκB (1, 3) and insulin (2,4) in sections of cultured human islets exposed for 44 hours to mediacontaining 5.5 mM (1, 2) or 33.3 mM glucose (3, 4). D, Doubleimmunostaining for NF-κB (p65) (1, 3) and insulin (2, 4) in human isletsexposed for 44 hours to media containing 5.5 mM (1, 2) or 33.3 mMglucose (3, 4). The arrows mark β-cell nuclei stained positive forNF-κB. 750-fold magnification. E, RT-PCR detection and quantification ofFas mRNA expression. Total RNA was isolated from human islets culturedfor 44 hours in a medium containing 5.5 or 33.3 mM glucose alone or inthe presence of IL-1Ra. In the Light Cycler quantitative PCR system, thelevel of Fas expression was normalized against GAPDH and the resultsexpressed as relative mRNA levels to control incubations at 5.5 mM.Results are shown ±SE for 6 independent experiments from 6 donors. *,P<0.05 relative to islets at 5.5 mM glucose; **, P<0.05 relative toislets at 33.3 mM glucose. F, Double immunostaining for Fas (1, 3, 5, 7,9, 11) and insulin (2, 4, 6, 8, 10, 12) in human islets exposed for 4days to media containing 5.5 mM glucose without (1, 2) and with IL-1βalone (3, 4) or with IL-1Ra (5, 6) or 33.3 mM glucose without (7, 8) andwith IL-1Ra (9, 10) or IL-1β (11, 12). 250-fold magnification. G, Humanislets were cultured for 4 days in 5.5 and 33.3 mM glucose alone or inthe presence of IL-1β and/or IL-1Ra or (H) with and without PDTC.Results are means±SE of the percent of TUNEL-positive β-cells. The meannumber of islets scored from each donor was 49 (range 35-63) for eachtreatment condition. Islets were isolated from five organ donors. *,P<0.01 relative to islets at 5.5 mM glucose; **, P<0.01 relative toislets at 33.3 mM glucose. +, P<0.01 relative to islets at 5.5 mMglucose plus IL-1β.

FIG. 5—Failure of glucose and IL-1β to induce iNOS mRNA expression inhuman islets.

RT-PCR analysis of iNOS expression by islets cultured for 44 hours in5.5 and 33.3 mM glucose or with IL-1β alone or in combination with IFNγ(positive control). GAPDH was used as control. One out of threeexperiments from three donors is shown.

FIG. 6—IL-1Ra and PDTC restore glucose-stimulated insulin secretion inhuman islets exposed to high glucose.

Islets were cultured on extracellular matrix-coated dishes for 4 days in5.5 and 33.3 mM glucose (control) or with IL-1Ra, PDTC, IL-1β incombination or individually. A, C, Basal and stimulated insulinsecretion denotes the amount secreted during successive 1 h incubationsat 3.3 and 16.7 mM glucose, respectively, following the 4 day cultureperiod. B, Insulin content. Data are represented as the mean of threeexperiments±SE from three separate donors. In each experiment, the datawere collected from three plates per treatment. *, P<0.01 relative toislets at 5.5 mM glucose; **, P<0.01 relative to islets at 33.3 mMglucose. ^(#), P<0.01 relative to islets at 5.5 mM glucose plus IL-1β.

FIG. 7—IL-1Ra is expressed by human islets and down-regulated in type 2diabetes.

Double immunostaining for IL-1Ra (A,C) or red (E) and insulin (B,D) orCD68 (F) in tissue sections of pancreases from a non-diabetic patient(A,B,E,F) and from a patient with type 2 diabetes (C,D). Magnification:×250. (G) Electron microscopy of cultured human islets doublegold-immunolabeled for insulin (small particles) and IL-1Ra (largeparticles).

FIG. 8—Leptin decreases β-cell production of IL-1Ra and induces IL-1βrelease in human islets.

(A) Double immunostaining for IL-1Ra (1,3,5) and insulin (2,4,6) insections of cultured human islets exposed for 48 h to media containing5.5 mM glucose alone (1,2), 33.3 mM glucose (3,4) or 5.5 mM glucose and10 nM leptin (5, 6). (B) Immunoblotting of IL-1Ra and actin. Humanislets cultured in a suspension with and without 10 or 500 nM leptinwere analyzed after 15 or 48 hours of incubation. The antibodies wereblotted on the same membrane after stripping. One out of threeexperiments from three donors is shown. Each experiment displayedsimilar results. (C) Secretion of IL-1Ra (1,3) and RT-PCR detection andquantification of IL-1Ra mRNA expression (2,4). Supernatants and totalRNA were obtained from human islets (1,2) and human blood monocytes(3,4) cultured for 15 and 48 hours in the presence of medium alone, 10or 500 nM leptin. In the LightCycler quantitative PCR system, the levelof IL-1Ra expression was normalized against tubulin, and the resultswere expressed as mRNA levels relative to control incubations. Data werecollected from three tubes per treatment of five separate experimentsfrom five islets donors and of three separate experiments from threeblood monocytes donors. Results are means±SE of percentage relative tocontrol incubations (100%, in absolute values for islets-IL-1Ra release:7.43±0.69 pg/islet/24 h and for monocytes-IL-1Ra release: 48.2±0.9ng/ml/24 h). *, P<0.01 compared to controls. (D) Secretion of IL-1β fromhuman islets (1) and human blood monocytes (2) cultured for 15 and 48hours in the presence of medium alone, 10 or 500 nM leptin. Data werecollected from three tubes per treatment of five separate experimentsfrom five islets donors and of three separate experiments from threemonocytes donors. Results are mean±SE of percentage relative to controlincubations (100%, in absolute values for islets-IL-1β release:0.11±0.04 pg/islet/24 h and for monocytes-IL-1Ra release: 228.2±44.3pg/ml/24 h). *, P<0.01 compared with controls. (E) Triple immunostainingfor IL-1β (1), insulin (2) and DNA fragmentation by the TUNEL assay inblack (3). The arrows mark a β-cell stained positive for IL-1β, insulinand the TUNEL reaction.

FIG. 9—Antagonizing IL-1Ra by siIL-1Ra induces β-cell apoptosis andimpairs β-cell function.

(A) RT-PCR detection and quantification of IL-1Ra mRNA expression. TotalRNA was isolated from human islets cultured for 48 hours in mediumcontaining 5.5 or 33.3 mM glucose alone (controls) or after transfectionwith 50 nM siIL-1Ra or with 50 nM of a scrambled RNA sequence(siScramble) with or without addition of 500 ng/ml exogenous rhIL-1Ra.In the Light Cycler quantitative PCR system, the level of IL-1Raexpression was normalized against tubulin and the results expressed asmRNA levels relative to control incubations at 5.5 mM. Results arepresented as means±SE of five independent experiments from five donors.*, P<0.001 compared to control islets at the same glucose concentration.(B) Immunoblotting of procaspase-3, activated caspase 3, IL-1Ra andactin. Human islets were cultured for 48 hours in medium containing 5.5mM glucose alone or after transfection with 50 nM siIL-1Ra or with 50 nMsiScramble. The antibodies were blotted on the same membrane afterstripping. One out of four experiments from four donors is shown. Eachexperiment displayed similar results. (C,D,E) Human islets were culturedon extracellular matrix coated dishes for 4 days in 5.5, 11.1 and 33.3mM glucose alone-(controls) or after transfection with 50 nM siIL-1Ra or50 nM siScramble with or without addition of 500 ng/ml exogenousrhIL-1Ra. (C) Triple immunostaining for IL-1Ra (1,4,7), insulin (2,5,8)and DNA fragmentation by the TUNEL assay in black (3,6,9) in isletsexposed to media containing 5.5 mM glucose alone (1, 2, 3) or withsiIL-1Ra (4, 5, 6) or with siIL-1Ra and rhIL-1Ra (7,8,9). The arrowsmark β-cell's nuclei stained positive for the TUNEL reaction. Note thatTUNEL-positive β-cells are negative for IL-1Ra. (D) Results are means±SEof percentage of TUNEL-positive β-cells relative to control incubationsat 5.5 mM glucose alone (100%, in absolute value: 0.33±0.03%TUNEL-positive β-cells). The mean number of islets scored from eachdonor was 44 (range 24-80) for each treatment condition. Islets wereisolated from five organ donors. ^(#), P<0.05 compared to control isletsat the same glucose concentration; *, P<0.05 compared with islets at 5.5mM glucose alone; ⁺, P<0.05 compared to siIL-1Ra transfected isletsalone at the same glucose concentration. (E) 1: basal and stimulatedinsulin secretion during successive 1-hour incubations at 3.3 (basal)and 16.7 (stimulated) mM glucose following the 4-day culture period. 2:insulin content. Data are presented as means±SE of three experimentsfrom three separate donors. In each experiment, the data were collectedfrom three plates per treatment. ^(#), P<0.05 compared with controlislets at the same glucose concentration; *, P<0.05 compared with isletsat 5.5 mM glucose alone; ⁺, P<0.05 compared to siIL-1Ra transfectedislets alone at the same glucose concentration.

FIG. 10—Leptin induces β-cell apoptosis and impairs β-cell function viaIL-1β-signaling.

(A) Human islets were cultured on extracellular matrix-coated dishes for4 days in 5.5 and 33.3 mM glucose (controls) or with 5 and 10 nM Leptin.Results are means±SE of percentage of TUNEL-positive β-cells relative tocontrol incubations at 5.5 mM glucose alone (100%, in absolute value:0.35±0.04 TUNEL-positive % β-cells). The mean number of islets scoredfrom each donor was 31 (range 21-45) for each treatment condition.Islets were isolated from five organ donors. *, P<0.01 compared withislets at 5.5 mM glucose alone; **, P<0.01 compared to islets at 33.3 mMglucose alone. (B) Immunoblotting of procaspase-3, activated caspase 3and actin. Human islets were cultured for 15 and 48 hours in mediumcontaining 5.5 mM glucose alone or with 10 or 500 nM leptin. Theantibodies were blotted on the same membrane after stripping. One out offour experiments from four donors is shown. Each experiment displayedsimilar results. (C,D) Islets were cultured on extracellularmatrix-coated dishes for 4 days in 5.5 and 33.3 mM glucose alone(controls) with or without 5 or 10 nM Leptin or with addition of 500ng/ml exogenous rhIL-1Ra. (C) 1: basal and stimulated insulin secretiondenote the amount secreted during successive 1-hour incubations at 3.3(basal) and 16.7 (stimulated) mM glucose following the 4-day cultureperiod. 2: insulin content. Data are represented as means±SE of threeexperiments from three separate donors. In each experiment, the datawere collected from three plates per treatment. *, P<0.01 compared toislets at 5.5 mM glucose alone; **, P<0.01 compared to islets at 33.3 mMglucose alone. (D) 1: results are means±SE of the percentage ofTUNEL-positive β-cells relative to control incubations at 5.5 mM glucosealone (100%, in absolute value: 0.37±0.06% TUNEL-positive β-cells). Themean number of islets scored from each donor was 29 (range 25-32) foreach treatment condition. 2: basal and stimulated insulin secretiondenotes the amount secreted during successive 1-hour incubations at 3.3(basal) and 16.7 (stimulated) mM glucose following the 4 day cultureperiod. 3: insulin content. Data are presented as means±SE of threeexperiments from three separate donors. In each experiment, the datawere collected from three plates per treatment. *, P<0.01 compared toislets at 5.5 mM glucose alone; ^(#), P<0.05 compared to leptin treatedislets alone.

DETAILED DESCRIPTION OF THE INVENTION

In advanced stages of type 2 diabetes, the β-cell function oftendegenerates to such a degree that insulin therapy becomes necessary. Theislet demise in humans with late type 2 diabetes is probably due to acombination of genetic and environmental components as well as tosecondary events including hyperglycemia-induced impaired β-cellfunction and apoptosis. The hallmark of type 1 diabetes is a specificand massive destruction of the β-cells, mostly by apoptosis. Althoughboth diseases have fundamental etiological differences, increasingevidence links both types of diabetes, and a significant fraction ofindividuals originally diagnosed with type 2 diabetes are cryptic type 1diabetics or evolve with time to a type 1 state, and exhibit anti-β-cellautoimmunity (41-44). Moreover, apoptotic cells can provoke an immuneresponse under the appropriate conditions, for example, when present inhigh enough numbers or exposed to cytokines such as IL-1β (45;46). Thus,induction of IL-1β by elevated glucose concentrations as shown in thepresent study or as part of an autoimmune response may connect type 2and type 1 diabetes.

Resident islet macrophages are fundamental in the development ofautoimmune diabetes (47;48). Activation of resident macrophages resultsin the expression and release of IL-1β (49). Recently, it has been shownthat viral replicative intermediate double-stranded RNA stimulates ratislet β-cell production of IL-1β as a mechanism by which viral infectionmay mediate β-cell damage in autoimmune diabetes (50). Interestingly,this same study revealed that, following such stimulation, rat β-cellsalso produce IL-1β converting enzyme, the enzyme required for activationof IL-1β. The results presented in this study indicate that humanβ-cells themselves are capable of producing IL-1β independently of anyviral infection or immune-mediated process, in response to glucose. Thefact that IL-1β producing β-cells were also detected in tissue sectionsof type 2 diabetic patients and in hyperglycemic but not euglycemicPsammomys obesus fully supports the in vitro observations and thecentral hypothesis of this study.

Diabetes-prone Psammomys obesus, when fed a high-energy diet, developdiabetes. As in some humans with type 2 diabetes, initialhyperinsulinemia in this animal model of diabetes progresses tohypoinsulinemia at later stages of the disease due to insufficientinsulin secretion and reduced pancreatic insulin reserve (51). In alongitudinal study, the present inventor and co-workers analyzed β-cellturnover during nutrition-induced diabetes (8). During the developmentof hyperglycemia an initial and transient increase in β-cell replicationoccurs, followed by a prolonged increase in the number of apoptoticβ-cells. The present inventor and co-workers now extend this study bythe observation of IL-1β expressing β-cells in parallel to insulindepletion in islets of hyperglycemic Psammomys obesus. Normalization ofplasma glucose by treatment of the animals with phlorizin preventedIL-1β expression and restored insulin expression in the islets,suggesting that in addition to its role as a mediator of glucose-inducedβ-cell apoptosis, IL-1β may participate in control of pancreatic insulinreserve.

To the best of the inventor's knowledge, iNOS can not be induced byIL-1β alone in human islets (52;53). Moreover, cytokine-induced Fasexpression is NO-independent (54;55). In line with these reports,glucose did not induce iNOS in the present study. Therefore, theIL-1β-mediated deleterious effects are probably NO-independent. However,it can not be excluded that NO is produced by β-cells in aniNOS-independent way, since IL-1β-induced nitrite has been demonstratedin the past (24;25;56), although this has not been observed by others(57).

At least 20 hours of exposure to high glucose are required to induceIL-1β, leading to NF-κB activation, Fas expression and β-cell death.This is in line with glucotoxic effects which appear only followingprolonged exposure to high glucose.

Inhibition of NF-κB activation by an adenoviral vector encoding for therepressor I-κB protects human islets from Fas-triggered apoptosis andresults in normal insulin response in the presence of IL-1β (56).Similarly, in purified rat β-cells, inhibition of cytokine-induced NF-κBactivation prevents β-cell apoptosis (59). Thus, the present findingthat glucose decreases I-κB expression and induces NF-κB activation viaIL-1β allows the prevention of glucotoxic effects by inhibition of NF-κBactivation.

So far, IL-1β production and release by islets was considered to belimited to type 1 diabetes. Here, the present inventor and co-workersdemonstrate that high concentrations of glucose induce IL-1β productionand secretion in human β-cells, leading to Fas receptor up-regulation,NF-κB activation, β-cell apoptosis and dysfunction. Moreover, thepresent inventor and co-workers observed IL-1β producing β-cells indiabetic patients and diabetic Psammomys obesus. The pathway by whichhyperglycemia causes impairment and loss of insulin producing cells thusshares features with immune-mediated processes. It follows that thepro-inflammatory cytokine IL-1β may be a crucial factor contributing toβ-cell glucotoxicity in the pathogenesis of type 2 diabetes. Theinventor believes that the new findings implicate that substances thatinhibit the action of the members of the IL-1β/NF-κB pathway can be usedto protect and preserve β-cell mass and function in prediabetic anddiabetic type 2 patients.

Thus, the present invention i.a. relates to the use of an Interleukin 1receptor antagonist (IL-1Ra) for the preparation of a medicament for thetreatment or prophylaxis of type 2 diabetes in a mammal, such as a dogor a human, in particular a human.

Thus, it should be understood that the present invention is applicablefor the treatment of patients already diagnosed as type 2 diabetespatients as well as for the prophylactic treatment of mammalspredisposed, e.g. genetically, environmentally, dietarily or sociallypredisposed, to type 2 diabetes.

When used herein, the term “type 2 diabetes” is defined as a metabolicdisorder characterized by hyperglycemia and abnormities in the glucose-protein- and lipid-metabolism. Type 2 diabetes is caused by insulinresistance which is not adequately compensated due to an insufficientβ-cell secretory capacity.

Interleukin 1 receptor antagonist (IL-1Ra) is a mature glycoprotein of152 amino acid (aa) residues. The protein has a native molecular weightof 25 kDa. When used herein, the terms “Interleukin 1 receptorantagonist” and “IL-1Ra”, and the like, are intended to encompasswild-type Interleukin 1 receptor antagonists as well as polypeptidesexhibiting substantially the same or improved biological activityrelative to the wild-type Interleukin 1 receptor antagonists. Suchpolypeptides include, without limitation, Interleukin 1 receptorantagonists that have been chemically modified, and Interleukin 1receptor antagonist variants into which specific amino acid sequencealterations have been introduced that modify the bioactivity of thepolypeptide. It further encompasses polypeptides with a slightlymodified amino acid sequence, e.g., polypeptides having a modifiedN-terminal end including N-terminal amino acid deletions or additions,and polypeptides having a modified C-terminal end including C-terminalamino acid deletions or additions.

IL-1Ra can be obtained from natural sources, e.g. by extraction andpurification from tissue or body fluids of mammals, such as humans,pork, goats, sheep, etc.; or by recombinant cell culture systems.

In one embodiment, the Interleukin 1 receptor antagonist (IL-1Ra) is arecombinant protein (rIL-1Ra). In a more specific embodiment, theInterleukin 1 receptor antagonist (IL-1Ra) is a recombinant humanprotein (rhIL-1Ra).

As an example of a suitable IL-1Ra should be mentioned the medicamentKineret® (anakinra) which is a recombinant, nonglycosylated form of thehuman interleukin 1 receptor antagonist (hIL-1Ra). Kineret® differs fromnative human IL-1Ra in that it has the addition of a single methionineresidue at its N-terminal end. Kineret® consists of 153 amino acids andhas a molecular weight of 17.3 kilodaltons. It is produced byrecombinant DNA technology using an E. coli bacterial expression system.Kineret® is supplied in single use 1 mL prefilled glass syringes with 27gauge needles as a sterile, clear, colorless-to-white, preservative-freesolution for daily subcutaneous (SC) administration. Each 1 mL prefilledglass syringe contains: 0.67 mL (100 mg) of anakinra in a solution(pH6.5) containing sodium citrate (1.29 mg), sodium chloride (5.48 mg),disodium EDTA (0.12 mg), and polysorbate 80 (0.70 mg) in Water forInjection, USP.

As another examples of a commercially available IL-1Ra can be mentionedthe recombinant porcine IL-1Ra provided by R&D Systems Inc.,Minneapolis, Minn., USA; Catalog No. 780-RA.

In an embodiment of the invention, the medicament comprises IL-1Ra incombination with pyrrolidinedithiocarbamate (PDTC).

A further embodiment of the invention relates to the use of the NF-κBinhibitor pyrrolidinedithiocarbamate (PDTC) for the preparation of amedicament for the treatment or prophylaxis of type 2 diabetes in amammal, such as a dog or a human, in particular a human.

The medicament comprising IL-1Ra and/or pyrrolidinedithiocarbamate(PDTC) is preferably administered parenterally, such as subcutaneously,intramuscularly or intravenously. General methods for the formulation ofIL-1Ra and/or pyrrolidinedithiocarbamate (PDTC) can be found inRemington: The Science and Practice of Pharmacy by Alfonso R. Gennaro(Editor), Lippincott, Williams & Wilkins; Dec. 15, 2000; ISBN:0683306472.

The medicament comprising IL-1Ra and/or pyrrolidinedithiocarbamate(PDTC) is preferably formulated with a pharmaceutically acceptablecarrier, e.g. adapted for parenteral administration, such assubcutaneous, intramuscular or intravenous administration.

The term “pharmaceutically acceptable carrier” is intended to encompassmedia generally acceptable for use in connection with the parenteraladministration of biologically active agents to a mammal such as ahuman. Pharmaceutically acceptable carriers are generally formulatedaccording to a number of factors well within the purview of the ordinaryskilled artisan to determine and account for, including withoutlimitation: the particular active drug substance(s) (IL-1Ra and/orpyrrolidinedithiocarbamate (PDTC)), its/their concentration, stabilityand intended bioavailability; the subject, its age, weight and generalcondition; and the intended route of administration of the composition,e.g. subcutaneous, intraperitoneal, intraveneous, or intramuscular.Typical pharmaceutically acceptable carriers used in parenteral drugadministration include, e.g., D5W (an aqueous solution containing 5%weight by volume of dextrose, and physiological saline. Pharmaceuticallyacceptable carriers can contain additional ingredients, e.g. those whichenhance the stability of the active drug substance(s) included, such aspreservatives and antioxidants.

A medicament for parenteral use (in particular subcutaneous,intraveneous, or intramuscular use) typically comprises 0.1 to 1000 mgof the active drug substance(s) per kg of the mammal's body.

The medicament for use in the context of the invention may comprise theactive drug substance(s) in the form of a sterile injection. To preparesuch a composition, the active drug substance(s) is/are dispersed ordissolved in a pharmaceutically acceptable carrier which convenientlymay comprise suspending, solubilising, stabilising, pH-adjusting and/ordispersing agents. Among acceptable carriers that may be employed arewater, water adjusted to a suitable pH by addition of an appropriateamount of an acid (e.g. hydrochloric acid), a base (e.g. sodiumhydroxide), or a suitable buffer, 1,3-butanediol, Ringer's solution andisotonic sodium chloride solution. The aqueous formulation may alsocontain one or more preservatives, e.g., methyl, ethyl or n-propylp-hydroxybenzoate.

In view of the above, it should be apparent that the present inventionalso provides a method of treating or prophylactically suppressing type2 diabetes, the method comprising administering to a mammal in needthereof a sufficient amount of an Interleukin 1 receptor antagonist(IL-1Ra). Furthermore, the present invention provides a method oftreating or prophylactically suppressing type 2 diabetes, the methodcomprising administering to a mammal in need thereof a sufficient amountof pyrrolidinedithiocarbamate (PDTC). The above embodiments andpreferences apply mutatis mutandis.

The applicability of IL-1Ra and/or pyrrolidinedithiocarbamate for thetreatment or prophylaxis of type 2 diabetes can be confirmed by animalmodel studies or clinical studies as described in the examples section.

Examples

Methods

Islet isolation and culture. Islets were isolated from pancreases ofeleven organ donors at the Department of Surgery, University of GenevaMedical Center, as described (32-34). The islet purity was >95%, asjudged by dithizone staining (if this degree of purity was not primarilyachieved by routine isolation, islets were handpicked). The donors, aged40-70 years, were heart-beating cadaver organ donors, and none had aprevious history of diabetes or metabolic disorders. For long-term invitro studies, the islets were cultured on extracellular matrix-coatedplates derived from bovine corneal endothelial cells (Novamed Ltd.,Jerusalem, Israel), allowing the cells to attach to the dishes andspread, preserving their functional integrity (7;35). Islets werecultured in CMRL 1066 medium containing 100 U/ml penicillin, 100 μg/mlstreptomycin and 10% fetal calf serum (Gibco, Gaithersburg, Md.),hereinafter referred to as culture medium. Two days after plating, whenmost islets were attached and began to flatten, the medium was changedto culture medium containing 5.5, 11.1 or 33.3 mM glucose. In someexperiments, islets were additionally cultured with 2 ng/ml recombinanthuman IL-1β, 1000 U/ml recombinant human IFNγ (ReproTech EC Ltd, London,UK), 500 ng/ml IL-1β receptor antagonist (IL-1Ra) (R&D Systems Inc.,Minneapolis, Minn.), 1 ng/ml membrane bound Fas-ligand (FasL) (36)(upstate biotechnology, Lake Placid, N.Y.) or with 100 μMpyrrolidinedithiocarbamate (PDTC; Sigma; 2 h per 2 d of culture).

Animals. Psammomys obesus of both sexes (age 2.0-3.5 months) from thediabetes-prone and diabetes-resistant lines of the Hebrew Universitycolonies were obtained from Harlan (Jerusalem, Israel). After weaning,diabetes-prone Psammomys obesus were maintained on a low-energy dietcontaining 2.38 kcal/g (Koffolk, Petach Tikva, Israel) until the startof the experiments, whereas diabetes-resistant Psammomys obesus weremaintained on a high-energy diet containing 2.93 kcal/g (WeizmannInstitute, Rehovot, Israel) to identify animals that develop diabetesand exclude them from the study (˜30-40% of the animals in thediabetes-resistant colony). All non-fasted animals with random bloodglucose concentrations <7.8 mmol/l (Glucometer Elite, Bayer Diagnostics,Elkart, Ind.) were considered nondiabetic. Diabetes-prone Psammomysobesus switched to a high-energy diet, received an injection of 0.4 g/kgphlorizin (Sigma) or solvent (40% propylene glycol) every 12 h and werekilled after 8 days. Psammomys obesus were anesthetized with ketamine(Ketalar, Park-Davis, Gwent, U.K.) and exsanguinated by cardiacpuncture. The pancreas was rapidly removed, and immersion-fixed in 10%phosphate-buffered formalin. The animal studies were approved by theInstitutional Animal Care and Use Committee of the Hebrew University andthe Hadassah Medical Organization.

Detection of IL-1β expressing β-cells. Pancreases from routinenecropsies and pancreases from Psammomys obesus were immersion-fixed informalin, followed by paraffin embedding. Sections were deparaffinizedand rehydrated, and endogenous peroxidase blocked by submersion in 0.3%H₂O₂ for 15 min. Sections were then incubated in methanol for 4 min.After washing with PBS, cultured islets and isolated β-cells were fixedin 4% paraformaldehyde (30 min, room temperature) followed bypermeabilisation with 0.5% triton X-100 (4 min, room temperature). Bothtissue sections and cultured cells were double-labeled for IL-1β andinsulin by 1 h exposure to 10% bovine serum albumin, followed byincubation (1 h, 37° C.) with mouse anti-IL-1β antibody (1:30 dilution,R&D Systems Inc.). Detection was performed using donkey anti-mouse Cy3conjugated antibody (1:100 dilution, Jackson). Subsequently, specimenswere incubated for 30 min at 37° C. with guinea pig anti-insulinantibody diluted 1:50 (Dako, Carpinteria, Calif.), followed by a 30 minincubation with a 1:20 dilution of fluorescein-conjugated rabbitanti-guinea pig antibody (Dako). For positive control of IL-1β staining,human mononuclear cells were isolated as described previously (37) andexposed for 2 h, 37° C. to 1 μg/ml lipopolysaccharide (Difco, Detroit,Ind.). Coverslips were air dried and mounted onto slides, fixed andpermeabilized for 5 minutes at room temperature in 50% acetone/methanoland stained for IL-1β as described.

For mRNA in situ hybridization of IL-1β, DNA templates were generated bypolymerase chain reaction with incorporation of a T3 or a T7 promotorinto the antisense or sense primer. The following primers were used: T35′AAGCGCGCAATTAACCCTCACTAAAGGGTCAGCACCTCTCAAGCAGAA3′ and T75′GGCCAGTAATTGTAATACGACTCACTATAGGGAGGCGGCCCTGAAAGGAGAGAGCTGA3′.Purification of PCR product was performed with Nucleospin Extract 2 in1™ (Machery-Nagel, Düren, Germany) according to the manufacturer'sinstruction. After phenol/chloroform purification, Digoxigenin-labeledRNA probes were prepared using RNA T7- and T3-polymerase and RNADigoxigenin labeling mix (Roche, Switzerland). Tissue sections weretreated with 20 μg/ml Proteinase K (Roche) and prehybridized for 2 hoursat 55° C. in hybridization buffer containing 50% formamide, 5× sodiumchloride-sodium citrate, 50 μg/ml salmon sperm (Sigma), 1× Denhart'ssolution, 250 μg/ml RNA Type IV from calf liver (Sigma). Thehybridization was performed overnight at 52° C. in 100 μl hybridizationbuffer containing 30 ng of Digoxigenin-labeled RNA probe. Sections werethen blocked with 5% milk powder at room temperature and incubated 1hour at 37° C. with anti-digoxigenin-rhodamine Fab fragment (20 μg/ml;Roche), followed by insulin immunostaining as described above.

After staining, samples were embedded in Kaiser's glycerol gelatin(Merck, Darmstadt, Germany) and analyzed by light and fluorescencemicroscopy (microscope Axiolab; Zeiss, Jena, Germany).

Western blot analysis. Islets were cultured in a culture medium innon-adherent plastic dishes. One day after isolation, the medium waschanged and groups of 200 islets were incubated for 44 hours in culturemedium containing 5.5 or 33.3 mM glucose without or with 2 ng/ml IL-1βor 500 ng/ml IL-1Ra. At the end of the incubations, islets were washedin PBS, suspended in 50 μl sample buffer containing 125 mM Tris-HCl,pH6.8, 4% SDS, 10% glycerol, 0.3% bromphenol blue, 1.8%β-mercaptoethanol and boiled for 5 min. Equivalent amounts of eachtreatment group were run on 15% SDS polyacrylamide gels. Proteins wereelectrically transferred to nitrocellulose filters and incubated withrabbit anti-Fas (C20, Santa Cruz Biotechnology Inc., Santa Cruz,Calif.), mouse anti-IL-1β (R&D Systems Inc), rabbit anti-IL-1β(recognizing precursor and cleaved forms of human IL-1β; Cell Signaling,Beverly, Mass.), mouse anti-NF-κB (p65) (Active Motif, Carlsbad,Calif.), mouse anti-NOS-2 (C-11, recognizing mouse, rat and human originof inducible nitric oxide synthase (iNOS), Santa Cruz BiotechnologyInc.) or mouse anti-actin (C-2; Santa Cruz Biotechnology Inc.)antibodies, followed by incubation with horseradish-peroxidase-linkedanti-mouse or anti-rabbit IgGs (Santa Cruz Biotechnology Inc.). Theemitted light was captured on X-ray film after adding Lumiglo reagent(Phototope-HRP Western blot detection kit; Biolabs, Beverly, Mass.). Asa marker, biotinylated protein molecular weight standard (Biolabs) wasrun in parallel. Between the incubations, nitrocellulose membranes werestripped for 30 min at 50° C. in 40 ml of a solution containing 280 μlβ-mercaptoethanol, 5 ml 0.5 M Tris-HCl, pH6.8 and 10% SDS, and then theywere washed for 1 h in Tris-buffered saline containing 0.1% Tween-20.Intensity of bands was analyzed using Multianalyst™ (Bio Rad,Laboratories inc., Hercules, Calif.).

NF-κB activation. Islets were cultured in a suspension as describedabove and washed with PBS. Activation of NF-κB complex was quantifiedwith an ELISA-based Kit, using attached oligonucleotides binding to aNF-κB consensus site and detected by an anti-p65 or p50 subunitantibody, according to the manufacturer's instructions (Trans-AM™ NFκB,Active Motif). In parallel, islets were fixed in Bouin solution for 15min, resuspended in 40 μl of 2% melted agarose in PBS (40° C.), followedby rapid centrifugation and paraffin embedding. Sections weredeparaffinized and rehydrated, endogenous peroxidase blocked bysubmersion in 0.3% H₂O₂ for 15 min and incubated in methanol for 4 min.Sections were incubated with mouse anti-NF-κB (p65) (1:50 dilution) orrabbit anti-IκB (1:50 dilution, C-21, Santa Cruz) antibodies, detectedby donkey anti-mouse or anti-rabbit Cy3 conjugated antibodies and doublestained for insulin as described above.

RNA extraction, RT-PCR and sequencing of RT-PCR product. Islets werecultured in a suspension as described above. Total RNA was extractedusing Rneasy mini kit (Qiagen, Basel, Switzerland) and RT-PCR wasperformed using the Superscript™ II Rnase H⁻ Reverse transcriptase KITand oligo-dT(24) (Life technologies, Gibco) according to theinstructions from the manufacturers. The primers were5′AAGCTGATGGCCCTAAACAG3′/5′AGGTGCATCGTGCACATAAG3′ (human IL-1β),5′GCATCTGGACCCTCCTACCT3′/5′CAGTCTGGTTCATCCCCATT3′ (human Fas) and5′acgtgcgttactccaccaaca3′/5′catagcggatgagctgagcatt3′ (human iNOS). Theconditions of the PCR amplification for IL-1β and Fas were: denaturation30 seconds at 94° C., annealing 30 seconds at 60° C. and elongation 30seconds at 72° C., followed for real time PCR by quantification 5seconds at 80° C., 45 cycles. The saturation of the PCR amplificationoccurred between 22 and 28 cycles. The size of the PCR amplificationproducts was 250 bp. The purified PCR products were sequenced to confirmamplification of the correct gene. The conditions of the PCRamplification for iNOS were: denaturation 30 seconds at 94° C.,annealing 40 seconds at 55° C. and elongation 30 seconds at 72° C., 35cycles. For quantitative analysis, we used the Light Cycler quantitativePCR system (Roche, Basel, Switzerland) and performed quantitative PCRwith a commercial kit (Light Cycler-DNA Master SYBR Green I; Roche). Theamount of Fas and IL-1β mRNA was standardized against GAPDH(5′AACAGCGACACCCACTCCTC3′/5′GGAGGGGAGATTCAGTGTGGT3′).

β-cell apoptosis. The free 3-OH strand breaks resulting from DNAdegradation were detected by the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) technique (38).After washing with PBS, cultured islets were fixed in 4%paraformaldehyde (30 min, room temperature) followed by permeabilisationwith 0.5% triton X-100 (4 min, room temperature). The TUNEL assay wasperformed according to the manufacturer's instructions (In Situ CellDeath Detection Kit, AP; Boehringer Mannheim, Germany). The preparationswere then rinsed with Tris-buffered saline and incubated (10 min, roomtemperature) with 5-bromo-4-chloro-indolyl phosphate/nitro bluetetrazolium liquid substrate system (Sigma). Then, islets were incubatedwith a guinea pig anti-insulin antibody as above, followed by detectionusing the streptavidin-biotin-peroxidase complex (Zymed). In parallel tothe TUNEL reaction, we used the DNA-binding dye propidium iodide (Sigma)to assess the effects of glucose on necrosis. Cultured islets werewashed with PBS (without paraformaldehyde-fixation), incubated for 10min on ice with 10 μg/ml propidium iodide in PBS, washed with PBS, andembedded in Dako fluorescent mounting medium (Dako). The samples wereimmediately evaluated by fluorescence microscopy for positively stainednecrotic nuclei.

Cytokine release. Cytokines release was evaluated in the culture mediumcollected before the termination of each experiment. The following kitswere used: human IL-1α, IL-1β, and IL-12 ELISA (R&D Systems Inc.), humanTNFα ELISA (Endogen, Boston, Mass.), human IFNγ ELISA (Gibco).

Insulin release and content. To determine acute insulin release inresponse to glucose stimulation, islets were washed in RPMI 1640 mediumwith 10% FCS and containing 3.3 mM glucose and pre-incubated for 1 h inthe same medium. The medium was then discarded and replaced with freshmedium containing 3.3 mM glucose for 1 h for basal secretion, followedby an additional 1 h incubation in medium containing 16.7 mM glucose.Incubates were collected and frozen for insulin assays. Then, isletswere washed with PBS and extracted with 0.18 N HCl in 70% ethanol for 24h at 4° C.; the acid-ethanol extracts were collected for determinationof insulin content. Insulin was determined by a human insulin RIA kit(CIS bio international, Gif-Sur-Yvette, France).

Evaluation and statistical analysis. Samples were evaluated in arandomized manner by a single investigator (K.M.) who was blinded to thetreatment conditions. Care was taken to score islets of similar size.Some larger islets did not completely spread and were several cellsthick. Such larger islets were excluded because a monolayer is aprerequisite for single cell evaluation. The mean surface of theevaluated islet-monolayers was 0.031±0.012 mm² and 0.029±0.011 mm² inislets cultured at 5.5 and 33.3 mM glucose, respectively (n. s.). Thus,the exclusion of larger islets occurred to a similar extent in each dishindependently of the treatment. Saisam™ software (Microvisioninstruments, Evry, France) was used to measure the areas. Data wereanalyzed by student's t test or by analysis of variance with aBonferroni correction for multiple group comparisons.

Results

Glucose induces IL-1β production and release in human islets. Humanislets were exposed to elevated glucose concentrations for 4 days.Measurement of the IL-1β released in the culture medium revealed a2.2-fold increase in islets cultured at 33.3 mM, relative to 5.5 mMglucose (FIG. 1A). To exclude a nonspecific effect of this highconcentration of D-glucose, osmolarity was corrected with 27.8 mML-glucose together with 5.5 mM D-glucose resulting in a similar releaseof IL-1β to that observed at 5.5 mM D-glucose alone. No IL-1β wasdetectable in unused culture medium. The specificity of IL-1β releasewas assessed by comparison with the release of other cytokines. Alimited amount of IL-1α and TNFα was found, but neither was regulated byglucose and no significant amount of IFNγ or IL-12 was detectable (Table1). The time-course of the effect of 33.3 mM glucose on IL-1β secretionrevealed a significant effect only after 20 hours of exposure to highglucose, persisting after 44 hours (FIG. 1B). Western blot analysis andquantitative RT-PCR measurement of IL-1β production in human isletsrevealed that elevated glucose concentration induces not onlyIL-1β-release but also IL-1β-protein and -RNA synthesis (FIGS. 1C & D).Note however, that Western blot analysis gave quite variable results interms of increased IL-1β in response to glucose. This may be due tovarying numbers of non-endocrine cells (including most notablymacrophages) in the different islet preparations. In the event of IL-1βproduction from such accompanying cells not being stimulated by glucose,it is found that the higher they are to total IL-1β production, thelower the expected glucose effect.

TABLE 1 Cytokines released by human islets cultured at low and highglucose concentrations Cytokine (pg/islet) 5.5 mM glucose 33.3 mMglucose IL-1α 0.71 ± 0.76 0.68 ± 0.67 IL-12 <0.06 <0.06 TNFα 5.68 ± 1.7 5.81 ± 1.96 IFN-γ <0.04 <0.04 Human islets were cultured for 4 days in5.5 or 33.3 mM glucose. Each number represents the mean of eightexperiments ± SE from eight separate donors.

Identification of the islet cellular source of glucose-dependent IL-1βproduction. We next identified the islet-cells producing IL-1β. Exposureof cultured human islets to 33.3 mM glucose for 4 days induced IL-1βexpression in clusters of β-cells, as determined bydouble-immunostaining of islets plated on extracellular matrix-coateddishes with anti-IL-1β and anti-insulin antibodies (for representativeimages from one experiment out of six from six donors see FIG. 2A-D. Ineach experiment, IL-1β positive β-cells were observed in islets culturedat 33.3 mM glucose). To exclude false-positive results due to IL-1βsecreted by other cells binding to β-cell membranes, islets were treatedwith IL-1Ra. Under these conditions, IL-1Ra should antagonize theinteraction of IL-1β with its surface receptors on β-cells.Co-incubation with IL-1Ra displayed similar results, confirming β-cellproduction of IL-1β.

IL-1β is not expressed in normal human pancreatic islets (28). However,based on the in vitro studies it was anticipated that it might beexpressed in islets of patients with type 2 diabetes, as a result ofhyperglycemia. Expression of IL-1β was therefore studied in sections ofpancreases from five poorly controlled type 2 diabetic patients, allwith documented fasting blood glucose>8 mM. Double-immunostaining of thepancreatic sections for IL-1β and insulin revealed localization of IL-1βto clusters of β-cells in all pancreases (22.5±3.4% of the islets perpancreas exhibited clusters of IL-1β expressing β-cells. Forrepresentative images, see FIG. 2G-H). The presence of IL-1β mRNAtranscripts was verified by in situ hybridization in β-cells of diabeticpatients (FIG. 2K-L). IL-1β expression could not be detected in β-cellsof non-diabetic controls (FIGS. 2E-F & I-J) and in the exocrinepancreas. A digoxigenin-labeled sense probe was used as control and gaveno signal (FIG. 2N).

β-cell expression of IL-1β during development of diabetes in Psammomysobesus is glucose-dependent. To examine whether induction of IL-1β invivo is also regulated by glucose, 3 diabetes-resistant and eightdiabetes-prone Psammomys obesus fed a low or high-energy diet werestudied and the hyperglycemic animals further treated with phlorizin,which corrects hyperglycemia by inhibiting renal tubular reabsorption ofglucose. No IL-1β expressing β-cells were observed in islets ofdiabetes-resistant and in fasted diabetes-prone Psammomys obesus (forrepresentative images see FIG. 3A-B). After 8 days of a high-energydiet, islets of severely hyperglycemic diabetes-prone Psammomys obesusexhibited IL-1β expression in most β-cells, which barely expressedinsulin (FIG. 3C-D). Normalization of blood glucose by injection ofphlorizin in animals fed a high-energy diet, restored insulin stores andprevented IL-1β expression (FIG. 3E-F).

IL-1β mediates glucose-induced NF-κB activation, Fas expression andβ-cell apoptosis. The functional role of glucose-induced IL-1β wastested using IL-1Ra as an inhibitor. In human islets, elevated glucoseconcentrations induced a 1.9-fold increase in NF-κB activity (FIG. 4A).This was prevented by IL-1Ra. The effect of glucose on NF-κB activationwas verified by Western blot analysis using an anti-NF-κB (p65)antibody, which exclusively recognizes the p65 subunit of the activenuclear form of the NF-κB transcription factor complex (39) (FIG. 4B).NF-κB is bound in the cytoplasm to inhibitory κB (IκB) proteins (40).Exposure of human islets to 33 mM glucose decreased IκB expression (FIG.4C) leading to nuclear apparition of NF-κB (p65) in β-cells (FIG. 4D).Glucose-dependent induction of Fas-receptor protein and mRNA were alsohindered by IL-1Ra (FIGS. 4B, E & F). Next, the ability of IL-1Ra toprotect the β-cells from glucose-induced apoptosis was evaluated.Exposure of human islets cultured on extracellular matrix dishes toelevated glucose concentrations increased the number of nucleidisplaying DNA fragmentation (TUNEL-positive) (FIG. 4G, H). Exposure ofislet cultures for 4 days to increasing glucose concentrations (from 5.5to 33.3 mM) did not, however, lead to propidium iodide uptake, therebyexcluding necrosis. IL-1Ra did not significantly change baselineapoptosis at 5.5 mM glucose. However, IL-1Ra protected the β-cells fromapoptosis induced by 33.3 mM glucose (FIG. 4G). Similarly, the NF-κBinhibitor PDTC inhibited glucose induced DNA fragmentation (FIG. 4H).Addition of exogenous FasL did not lead to a significant increase ofβ-cell death (2.8±0.4-fold increase of TUNEL-positive β-cells in 33.3 mMglucose alone vs. 3.0±0.4-fold increase in 33 mM glucose+exogenous FasL,as compared to control at 5.5 mM glucose). Finally, it was examinedwhether high glucose induces iNOS expression. Neither 33 mM glucose norIL-1β alone induced iNOS mRNA (FIG. 5) or protein expression.

IL-1Ra and PDTC improve impaired β-cell function due to IL-1β mediated“glucotoxicity”. Chronic exposure of human islets to 33.3 mM glucose orIL1β for 4 days abolished acute glucose-stimulated insulin release(FIGS. 6A & C). Co-incubation with IL-1Ra or PDTC partially restoredsuch glucose stimulation. Insulin content of islets cultured at highglucose decreased compared to control (5.5 mM glucose), and remainedunaffected by IL-1Ra (FIG. 6B).

Animal Study. Treatment of Psammomys obesus, an Animal Model of Type 2Diabetes Mellitus, with Interleukin-1 Receptor Antagonist. (SuggestedStudy)

The therapeutic potential of exogenous administration of IL-1Ra toprevent the decline of β-cell mass observed in type 2 diabetes patientswill be tested in Psammomys obesus. The gerbil Psammomys obesus showsinsulin resistance and develops diet-induced obesity-linked diabetes,initially associated with hyperinsulinemia, and gradually progressing tohypoinsulinemia and severe hyperglycemia. This is accompanied by atransient increase in beta-cell proliferative activity and by aprolonged increase in the rate of beta-cell death, culminating indisruption of islet architecture (8). Because of the obvioussimilarities, it serves as a convenient model for human type 2 diabetes.We will explore whether treatment with IL-1Ra may preventhyperglycemia-induced β-cell apoptosis and impaired proliferation inpancreatic islets of Psammomys obesus during development of diabetes. Asa part of an ongoing collaboration with the group of N. Kaiser (HadassahUniversity Hospital, Jerusalem), 10 mg/Kg body wt/d of IL-1Ra (Kineret®)will be injected intraperitoneally to Psammomys obesus. The study willcomprise 3 groups of diabetes-prone Psammomys:

Group 1, Psammomys maintained on low-energy diet.

Group 2, Psammomys treated with IL-1Ra and switched from low-energy tohigh-energy diet to induce diabetes.

Group 3, Psammomys treated with solvent and switched from low-energy tohigh-energy diet to induce diabetes.

Four animals of each group will be sacrificed on days 4, 7, 14, 21 and28 of the study. Upon sacrifice, blood will be collected and used formeasurement of plasma glucose, insulin and triglycerides. The pancreaswill be removed and the head-portion frozen at −70° C. for subsequentdetermination of insulin content. The remaining tail-part will be fixedin 10% phosphate buffered formalin, followed by standard paraffinembedding. Sequential sections will be analyzed for expression of Fas,IL-1β, insulin as well as for β-cell proliferation and apoptosis. Theproposed number of animals and the duration of the treatment are basedon our previous studies with Psammomys (8).

Anticipated Conclusion

Assuming that glucotoxicity will be completely blocked by IL-1Ra, thefollowing improvements can be expected in IL-1Ra treated animals ascompared to placebo-treated:

Prevention or delay of diabetes onset

Protection from hyperglycemia-induced β-cell apoptosis, impairedproliferation and decreased β-cell mass.

Normalisation of pancreatic insulin content.

Clinical Study. Treatment of Patients with Type 2 Diabetes Mellitus withInterleukin-1 Receptor Antagonist. (Suggested Study)

72 patients will be randomised according to a double-blind,placebo-controlled protocol in which half of the patients are treatedwith IL-1Ra, the other half with saline. The treatment period will last13 weeks. This time-period should be sufficient for reversal offunctional glucotoxicity (61) and feasible in terms of patientcompliance. Whether 13 weeks of treatment will be sufficient to makesignificant changes in β-cell mass in unpredictable. However, blockingβ-cell apoptosis, while new islet formation and β-cell replication arenormal (62), may initiate enlargement of β-cell mass, which may progressbeyond the treatment period. Patient evaluation will be performed atstart and after 4, 13, 26, 39 and 52 weeks. Following 13 weeks, patientswith a fasting plasma glucose levels>8 mM or with a glycosylatedhemoglobin level (HbA1c)>8% will be treated with insulin. Insulintreatment will not be initiated earlier to avoid interference withpossible effects of insulin on primary outcome in the period where thelargest effect of IL-1Ra are expected. To assess effects of IL-1Ra oninsulin sensitivity, a subset of 40 patients (20 IL-1Ra- and 20placebo-treated) will undergo an euglycemic-hyperinsulinemic clamp aswell as a muscle and fat biopsy at start and after the end of treatment(13 weeks).

Inclusion Criteria:

-   -   Age>30    -   Diabetes mellitus Type 2 (American Diabetes Association        criteria) of at least 3 months duration and treated solely with        diet and exercise and/or oral antidiabetic drugs.    -   HbA1c>8%    -   Body-mass index (BMI)>27

Exclusion Criteria

-   -   Positive GAD 2 or IA-2 antibodies    -   HbA1c>12%, polyuria and thirst (exclusion of severely        decompensated patients)    -   Current treatment with insulin    -   Established anti-inflammatory therapy    -   CRP>30 mg/dl, fever, current treatment with antibiotics, or        chronic granulomatous infections (e.g. tuberculosis) in the        history or on a screening chest X-ray.    -   Neutropenia or anemia (leucocyte count<2.0×10⁹/l, haemoglobin<11        g/dl for males or <10 g/dl for females)    -   Pregnancy or breast-feeding    -   Severe liver or renal disease (AST or ALT>3 times the upper        limit of normal laboratory range, serum creatinine>130 μM)    -   Ongoing malignant neoplasm    -   Use of any investigational drug within 30 days of enrollment        into the study or within 5 half-lives of the investigational        drug (whichever is the longer)

Primary Endpoints:

-   -   Stimulated C-peptide and insulin (see below)    -   HbA1c    -   Fasting plasma glucose (FPG)

Secondary Endpoints:

-   -   Insulin requirement    -   Serum cytokines levels, CRP    -   Insulin secretion and Insulin-sensitivity index derived from an        OGTT with insulin and glucose measurements.    -   In a subgroup of patients, insulin-sensitivity assessed by clamp        techniques as well as by muscle and fat biopsies.

Patient Evaluation

Patients will be evaluated as follows:

-   -   Physical examination including Body Mass Index, Waist to Hip        Ratio, blood pressure (standing and supine), heart rate    -   Blood samples for determination of HbA1c, lipid profile        including free fatty acids, HDL- and LDL-cholesterol, IL-1β,        IL-1Ra, IL-6, TNFα, CRP, sodium, potassium, creatinine, AST,        ALT, and hematogramm.    -   24 h urine collection for albuminuria and creatinine clearance        (only baseline and end of study).    -   Ophthalmologic examination including stereoscopic fundus        photography (only baseline and end of study)    -   Standard oral glucose-tolerance-test (OGTT) with measurement of        plasma blood glucose, insulin and C-peptide at 0, 30, 60, 90 and        120 min. At 120 min, 0.3 g/kg glucose+0.5 mg glucagon+5 g        arginine will be injected intravenously followed by measurement        of plasma blood glucose and insulin at 0, 3, 6, 9 and 12 min.    -   Weekly full blood glucose profile performed at home by the        patient.    -   Euglycemic-hyperinsulinemic clamp and biopsies: a subset of 40        patients (20 IL-1Ra- and 20 placebo-treated) will undergo an        euglycemic-hyperinsulinemic clamp as well as a muscle and fat        biopsy. This technique is routinely used in the group of A. Vaag        at the SDC (63-65). M. Faulenbach will learn the technique at        the SDC and introduce it at the USZ. Polyethylene catheters will        be placed in the antecubital vein for infusion and in the        contralateral dorsal hand or antecubital vein for blood        sampling. This “sampling” hand will be placed in a heated        Plexiglas box to ensure arterialization of the venous blood        sample. After an initial 40-min basal period, a        primed-continuous insulin infusion (40 mU·m⁻²·min⁻¹) will be        initiated and continued for 3 h. Basal and insulin stimulated        steady state periods will be defined as the last 30 min of the        40 min basal state period and the last 30 min period of the 3 h        clamp period. A variable infusion of glucose (180 g/l) will        maintain euglycaemia during insulin infusion. Plasma glucose        concentration will be monitored every 5 to 10 min during the        basal and clamp periods using an automated glucose oxidation        method. Blood samples will be drawn for measurements of insulin        every 10 to 30 min during the basal and clamp steady state        periods. Needle biopsies will be obtained in the basal state        (time 0 min) from the vastus lateralis muscle and from the        subcutaneous fat of the same region as well as from the        abdominal region. The biopsies will be immediately frozen in        liquid nitrogen and stored at −80° C. until analyzed for        expression of cytokines (e. g. TNFα, IL-1α and β, IL-1Ra, IL-6,        adiponectin and leptin) as well as for other genes and proteins        of potential importance for insulin action.    -   The patients will be instructed to abstain from strenuous        physical activity for 24 h and to fast for 9-10 h before both        tests (OGTT and clamp studies). They should receive an injection        of the study medication on study days but not other antidiabetic        medications. The clamp will follow the OGTT, with a separation        of 2 to 7 days. Study medication will be continued until the end        of all assessments.

Basic Medication:

Any change of patients' current therapy during the study should beavoided.

Study Medication:

Anakinra (Kineret®) is the recombinant, nonglycosylated form of theIL-1Ra with an N-terminal methionine and is produced by Amgen. Bycompetitively binding to the interleukin-1 type I receptor, anakinrainhibits the activity of IL-1. FDA approval for a 100 mg dose viasubcutaneous injection for 6 months occurred in November 2001. Anakinrahas a half-life of 4-6 hours and a maximum plasma concentration at 3-7hours after subcutaneous administration of 1-2 mg/kg.

Anakinra will be given every 24 h in a single morning dose of 100 mg.Anakinra or placebo (saline) will be injected subcutaneously into theskin of the abdomen or upper thighs. The study nurse will instruct thepatients how to perform the injections by themselves. One physician willalways be available throughout 24 h for health or any other problems.

Ethical Considerations:

-   -   Adverse reactions

Injection site reactions, characterized by erythema, ecchymosis,pruritus, inflammation, pain, and swelling, are the most commonlyreported adverse effects of subcutaneous treatment in rheumatoidarthritis patients. Reactions were usually mild and transient, resolvingwithin several weeks; however, discontinuation of treatment has beenrequired in up to 5% of patients. In clinical trials of rheumatoidarthritis, neutropenia with absolute neutrophil count 1×10⁹/l or lessdeveloped in 0.3% of patients who received IL-1Ra in placebo-controlledtrials. Infections occurred in 40% of patients who received IL-1Ra and35% of patients who received placebo (n. s.). Serious infectionsoccurred in 1.8% of the IL-1Ra group and 0.6% of the placebo groupduring a 6-month period (n. s.). Serious infections were primarilybacterial (cellulitis, pneumonia, bone and joint infections) rather thanopportunistic, fungal, or viral.

-   -   Invasive investigations (clamp and biopsies)

The euglycemic-hyperinsulinemic clamp is the gold standard technique toassess insulin sensitivity. All the necessary catheters involve onlyperipheral veins. Blood glucose will be monitored every 5 to 10 min,which should be sufficient to avoid hypoglycemia.

A muscle and fat biopsy will give unique insight into the effect ofIL-1Ra on insulin-sensitive tissues. Local anesthesia will avoid anysignificant discomfort. The size of the samples will be maximum 0.5 cm³.

-   -   Injections of study medication

Most of the participating patients will eventually have to injectinsulin. Therefore, the burden to learn and get used toauto-subcutaneous injection will be beneficial for most of them in thelong-term.

-   -   Delay of instituting insulin therapy until 13 weeks

Since severely hyperglycemic patients are excluded from the study, apossible delay of 13 weeks until they start using insulin, should notsignificantly increase the risk of developing long-term complications ofdiabetes.

-   -   Breaking of criteria

Patients have the right to withdraw at any given time from the studywithout giving a reason. The study doctor can interrupt or end thetherapy if any side effects arise or an unexpected course of the therapyis detected, or if it would be in the interest of the patient. In thissituation, the study doctor decides on the following therapeuticmeasures. Withdrawal from the study will not affect further care.

-   -   Informed-consent

A written informed-consent form will be obtained from each patient.

Statistics

Based on a power calculation including an expected 30% change instimulated insulin, an SD of 50%, a power (1-β) of 80% and asignificance of α=0.05, it is estimated that a sample size of 72(placebo:IL-1Ra 1:1) will be required.

Student's two tailed t-test will be used for comparison of means.Multiple measurements obtained over time will be analyzed byone-way-analysis of variance (ANOVA) for repeated measures.Matched-pairs (comparison of baseline measurements and follow-up) willbe tested by the Wilcoxon test.

Blinding and randomization will be performed by the Kantonsapotheke,Zurich.

Anticipated Conclusion

Assuming that glucotoxicity will be completely blocked by IL-1Ra, thefollowing improvements can be expected in IL-1Ra treated patients ascompared to baseline or placebo-treated patients:

-   -   60% (or higher) increase in stimulated C-peptide and insulin        levels.    -   Improvement of HbA1c: depending on baseline HbA1c, a decrease of        HbA1c of 1% (baseline 8%) to 4% (baseline 12%).    -   Fasting plasma glucose (FPG): depending on baseline FPG, a        decrease of FPG by 13% (baseline 8 mM glucose) to 27% (baseline        15 mM glucose).    -   No insulin requirement in the IL-1Ra-treated group versus 0.8        IU/Kg insulin in the placebo-treated.    -   60% (or higher) increase in insulin-sensitivity.    -   Normalisation of serum cytokines and CRP levels.

Further Study of Role of IL-1Ra in Type 2 Diabetes

The invention is further supported by the result of the following studyof the molecular link between obesity and type 2 diabetes, in particularthe role of IL-1Ra.

Obesity is associated with an increased risk of diabetes, but themechanisms of this progression are unclear. Most obese people areinsulin resistant and have to adapt by increasing insulin secretion. Theβ-cell mass itself is the major factor in the amount of insulin that canbe secreted and β-cell mass increases during obesity (66;62). Inindividuals who lose the ability to produce sufficient quantities ofinsulin to maintain normoglycemia in the face of insulin resistance,type 2 diabetes mellitus manifests (67). Increasing evidence suggeststhat a progressive decrease in β-cell mass, and not only β-cellfunction, contributes to this (62;68;69). The deficit of β-cell mass inthe pathophysiology of type 2 diabetes seems to be due to increasedβ-cell apoptosis (62;70). Possible mediators of the process of β-celldestruction are increased serum concentrations of cell nutrients.Indeed, increased free fatty acid (FFA) levels per se are known to betoxic for β-cells, leading to the concept of lipotoxicity (12;71-75).However, not all obese individuals or pre-type 2 diabetes patientsexhibit dyslipidemia. Thus, lipotoxicity may play an important role inthe process of β-cell destruction but probably does not act alone.Elevated glucose concentrations induce β-cell apoptosis in culturedislets from diabetes-prone Psammomys obesus, an animal model of type 2diabetes (70), in human islets (9-10;76-77) and at higher concentrationsin rodent islets (11-12). In human islets, glucose-induced β-cellapoptosis and dysfunction are mediated by β-cell production andsecretion of IL-1β (76). Probably already in the pre-type 2 diabeticstage, insulin resistance diminishes glucose uptake, resulting intransient post-prandial hyperglycemic excursions, supporting theglucotoxicity hypothesis (3-7;35;78-80). This transient hyperglycemiacould act on the β-cells even before diabetes manifests itself or at thevery early stages of the disease, but it is unclear whether this issufficient to cause progression from obesity to diabetes.

Leptin is expressed primarily in the adipose tissue and thereforerepresents the most obvious exponent of the adipocyte (81). β-cellsexpress leptin receptors, leptin inhibits insulin secretion in vivo andin vitro (82-85). In rodent islets, leptin induces β-cell proliferationand protects from FFA-induced β-cell apoptosis (86-89). However, theeffect of leptin on human β-cell apoptosis is not known. This is ofparticular interest, because human β-cells often respond differentlythan rodent islets with respect to stimuli of apoptosis. For example,increasing ambient glucose concentrations from physiologic 5.5 mM to11.1 mM decreases the rate of β-cell apoptosis in rodents islets,whereas it induces apoptosis in human islets (9;11).

Interleukin-1 receptor antagonist (IL-1Ra) is an anti-inflammatorycytokine and naturally occurring antagonist of IL-1α and β (90;91).Similarly to IL-1β, IL-1Ra binds to type 1 IL-1 receptor but it lacks asecond binding domain and therefore does not recruit the IL-1 receptoraccessory protein, the second chain of the receptor complex. Three formsof IL-1Ra have been described, two of them are intracellular proteins(icIL-1Ra I and II) and one is secreted. The function of icIL-1Ra is notknown (93). Exogenous recombinant human (rh) IL-1Ra protects culturedhuman islets from glucose-induced, IL-1β-mediated, β-cell apoptosis andimproves β-cell function (76). Interestingly, leptin induces secretionof IL-1Ra in monocytes (93). However, expression and regulation ofIL-1Ra in human islets have not been investigated.

Therefore, IL-1Ra in human pancreatic β-cells of non-diabetic anddiabetic patients was studied and its regulation by leptin wasidentified. Effects of leptin on IL-1β production and on human β-cellsurvival and function, partly mediated by changes in IL-1Ra expression,were also explored.

Methods

Islet isolation and cell culture. Islets were isolated from pancreasesof ten organ donors at the Department of Surgery, University of GenevaMedical Center, as described (32-34). The islet purity was >950/%, asjudged by dithizone staining (if this degree of purity was not primarilyachieved by routine isolation, islets were handpicked). The donors, aged40-65 years, were heart-beating cadaver organ donors, and none had aprevious history of diabetes or metabolic disorders. For long-term invitro studies, the islets were cultured on extracellular matrix-coatedplates derived from bovine corneal endothelial cells (Novamed Ltd.,Jerusalem, Israel), allowing the cells to attach to the dishes andspread, preserving their functional integrity (7;35). Islets werecultured in CMRL 1066 medium containing 100 U/ml penicillin, 100 μg/mlstreptomycin and 10% fetal calf serum (Invitrogen Corporation, Paisley,U.K.), hereafter referred to as culture medium. Two days after plating,when most islets were attached and began to flatten, the medium waschanged to culture medium containing 5.5, 11.1 or 33.3 mM glucose. Insome experiments, islets were additionally cultured with 5, 10 or 500 nMrh leptin (PeproTech, London, UK), 500 ng/ml rh IL-1Ra (R&D SystemsInc., Minneapolis, Minn.) or 50 nM small interfering RNA (siRNA) asdescribed below.

Human blood monocytes were isolated as described previously (37),cultured in RPMI 1640 (Invitrogen) containing 100 U/ml penicillin, 100μg/ml streptomycin and 10% fetal calf serum and exposed to 10 or 500 nMleptin.

RNA interference. RNAs of 21 nucleotides, designed to target humanIL-1Ra (5′AUCUGCAGAGGCCUCCGCAtt3′/5′UGCGGAGGCCUCUGCAGAUtt3′) andscrambled siRNA were synthesized by Ambion (Austin, Tex.). siRNAs weretransfected using SiPortAmine™ according to the manufacturer'sinstructions (Ambion).

Detection of IL-1Ra and IL-1β expressing cells. Pancreases from routinenecropsies were immersion-fixed in formalin, followed by paraffinembedding. In parallel, isolated human were cultured in a suspension for48 hours and exposed to 10 nM leptin or 33.3 mM glucose, islets werefixed in Bouin solution for 15 min, resuspended in 40 μl of 2% meltedagarose in PBS (40° C.), followed by rapid centrifugation and paraffinembedding. Sections were deparaffinized, rehydrated and then incubatedin methanol for 4 min. Human islets cultured on extracellular matrix for4 days were washed in PBS, fixed in 4% paraformaldehyde (30 min, roomtemperature) followed by permeabilization with 0.5% triton X-100 (4 min,room temperature). Pancreas and islet sections and cultured cells weredouble-labeled for IL-1Ra or IL-1β and insulin, CD68, glucagon orsomatostatin by 1 h of exposure to 10% bovine serum albumin followed byincubation (1 h, 37° C.) with biotinylated goat anti-IL-1Ra or mouseanti-IL-1β antibodies (R&D Systems Inc.). Detection was performed usingcy3-(Jackson, ImMunoResearch Laboratories, West Grove, Pa.) orcy2-(Transduction Lab., Lexington, Ky.) conjugated streptavidin.Subsequently, specimens were incubated for 30 min at 37° C. withguinea-pig (Dako, Carpinteria, Calif.) or mouse (Sigma) anti-insulin,rabbit anti-somatostatin (Dako), rabbit anti-glucagon (Dako) or mouseanti-CD68 (Biosource, Nivelles, Belgium) antibodies, followed by a 30min incubation with FITC-conjugated rabbit anti-guinea pig (Dako),cy3-conjugated donkey anti-mouse, cy3-conjugated donkey anti-rabbit orcy2-conjugated donkey anti-mouse antibodies (Jackson).

Electron microscopy. Human islets maintained in culture medium innonadherent plastic dishes were fixed in a solution containing freshlymade 2.5% paraformaldehyde, 0.1% glutardialdehyde and 0.01% picric acidfor about 4 h at room temperature. Thereafter, specimens were dehydratedin an ascending series of ethanol and routinely embedded in LR White(Polysciences, Warrington, Pa., USA). Ultrathin sections were treatedwith 50 mM gelatine in 10 ml PBS containing 0.2 g BSA and rinsed in PBS.Sections were incubated with guinea-pig anti-porcine insulin (code:A564; 1:400; Dako) overnight at room temperature, washed in PBS,followed by anti guinea pig IgG conjugated with a gold-5 nm complex(1:50, Amersham Int., Little Chalfont, UK) for 30 min. Thereafter,sections were rinsed in double-distilled water, air dried, and incubatedwith the biotinylated goat anti-IL-1Ra antibody as described above,washed in PBS followed by incubation with a streptavidin-gold-5 nmcomplex (1:50, Amersham) for 1 h at room temperature. Sections werecounterstained with uranyl acetate for 4 min and examined andphotographed with a Philips EM 420 electron microscope.

Western blot analysis. Human islets were maintained in culture medium innonadherent plastic dishes. One day after isolation, the medium waschanged and groups of 200 islets were incubated in culture mediumcontaining 5.5 glucose with or without 10 or 500 nM leptin or 50 nMsiRNA. At the end of the incubations, islets were washed in PBS,suspended in 40 μl sample buffer containing 125 mM Tris-HCl (pH6.8), 4%SDS, 10% glycerol, 0.3% bromphenol blue, 1.8% β-mercaptoethanol andboiled for 5 min. Equivalent amounts of each treatment group were run on15% SDS polyacrylamide gels. Proteins were electrically transferred tonitrocellulose filters and incubated with biotinylated goat anti-IL-1Ra,mouse anti-caspase 3 (which binds to both, procaspase-3 and activatedcaspase-3; Pharmingen, San Diego, Calif.), or mouse anti-actin (C-2;Santa Cruz Biotechnology Inc., Santa Cruz, Calif.) antibodies, followedby incubation with horseradish-peroxidase-linked anti-mouse (Santa CruzBiotechnology Inc.) or streptavidin (Jackson). The emitted light wascaptured on X-ray film after adding Lumi-Light Western BlottingSubstrate (Roche, Basel, Switzerland). As a marker, KaleidoscopePrestained Standards (Bio Rad, Laboratories inc., Hercules, Calif.) wasrun in parallel. Between incubations, nitrocellulose membranes werestripped for 30 min at 50° C. in 40 ml of a solution containing 280 μlβ-mercaptoethanol, 5 ml 0.5 M Tris-HCl (pH6.8) and 10% SDS, and thenwashed for 1 h in Tris-buffered saline containing 0.1% Tween-20.

RNA extraction and RT-PCR. Islets were cultured in a suspension asdescribed above. Total RNA was extracted using Rneasy mini kit (Qiagen,Basel, Switzerland) and RT-PCR was performed using the Superscript™ IIRnase H⁻ Reverse transcriptase kIT and oligo (dT) (7) (Lifetechnologies, Gaithersburg, Md., USA) according to the instructions fromthe manufacturers. The following primers were used:5′ACTGAGGACCAGCCATTGAG3′/5′AGGTGGAATGAGGGAGGAAG3′ (human IL-1Ra) and5′AAGCTGATGGCCCTAAACAG3′/5′AGGTGCATCGTGCACATAAG3′ (human IL-1β). PCRconditions were: denaturation for 30 seconds at 94° C., annealing for 30seconds at 60° C., and elongation for 30 seconds at 72° C., followed forreal time PCR by quantification for 5 seconds at 82° C.; repetition for40 cycles. Saturation of PCR product occurred between 19 and 33 cycles.The size of the PCR amplification product was 250 bp. For quantitativeanalysis, we used the LightCycler quantitative PCR system (Roche) andperformed quantitative PCR with a commercial kit (LightCycler-DNA MasterSYBR Green I; Roche). The amounts of IL-1Ra mRNA were standardizedagainst α-Tubulin (5′AGAGTCGCGCTGTAAGAAGC3′/5′TGGTCTTGTCACTTGGCATC3′)and GAPDH (5′AACAGCGACACCCACTCCTC3′/5′GGAGGGGAGATTCAGTGTGGT3′) withsimilar results.

β-cell apoptosis. The free 3′-OH strand breaks resulting from DNAdegradation were detected using the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) technique asdescribed in detail (70;76;77). Thereafter, islets were incubated withguinea pig anti-insulin antibody as above, followed by detection usingthe streptavidin-biotin-peroxidase complex (Zymed Laboratories Inc.,South San Francisco, Calif., USA). In parallel to the TUNEL reaction,apoptosis was confirmed by detection of caspase 3 activation asdescribed above (Western blot analysis).

Cytokine release. Cytokine release was evaluated in the culture mediumcollected before the termination of each experiment using human IL-1βand IL-1Ra ELISA kits (R&D Systems Inc.).

Insulin release and content. To determine acute insulin release inresponse to glucose stimulation, islets were washed in Kreb's ringerbicarbonate buffer (KRB-Hepes, pH7.4: 4.8 mM KCl, 134 mM NaCl, 5 mMNaHCO₃, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 1 mM CaCl₂, 0.5% BSA, 10 mM HEPES)containing 3.3 mM glucose and pre-incubated for 30 min in the samebuffer. The KRB was then discarded and replaced by fresh buffercontaining 3.3 mM glucose for 1 h for basal secretion, followed by anadditional 1 h of incubation in KRB containing 16.7 mM glucose.Supernatants were collected and frozen for insulin assays. Thereafter,islets were washed with PBS and extracted with 0.18 N HCl in 70% ethanolfor 24 h at 4° C. The acid-ethanol extracts were collected fordetermination of insulin content. Insulin was determined by a humaninsulin RIA kit (CIS bio international, Gif-Sur-Yvette, France).

Evaluation and statistical analysis. Samples were evaluated in arandomized manner by a single investigator (K.M.) who was blinded to thetreatment conditions. Care was taken to score islets of similar size.Some larger islets did not completely spread and were several cellsthick. Such larger islets were excluded because a monolayer is aprerequisite for single cell evaluation. The mean surface of theevaluated islet-monolayers was determined previously in islets culturedat 5.5 and 33.3 mM glucose, respectively, and found to be similar(76;77). Thus, the exclusion of larger islets occurred to a similarextent in each dish independently of the treatment. Data were analyzedby student's t test or by analysis of variance with a Bonferronicorrection for multiple group comparisons.

Results

IL-1Ra is expressed by human pancreatic β-cells and downregulated intype 2 diabetic patients. Immunodetection of human pancreatic sectionsfrom non-diabetic patients revealed the presence of IL-1Ra localized inthe β-cells (FIGS. 7A & B). Additionally, some non-β-cells expressedIL-1Ra and were identified as resident macrophages (CD68 positive; FIGS.7E & F). No specific staining for IL-1Ra was observed in α and δ cells,as assessed by double immunostaining with anti-IL-1Ra and anti-glucagonor anti-somatostatin antibodies, respectively. IL-1Ra was alsoundetectable in the exocrine tissue. Expression of IL-1Ra by the β-cellitself was confirmed by electron microscopy of sections from isolatedhuman islets (FIG. 7G). Next, expression of IL-1Ra in sections ofpancreases from six poorly controlled type 2 diabetic patients wasstudied, all with documented fasting blood glucose>8 mM (Table 2). Inall pancreases, IL-1Ra protein expression was decreased as compared tothe non-diabetic controls (for representative images see FIGS. 7C & D).

TABLE 2 No age sex glucose (mM) diagnosis diabetic 1 90 f 8.9 COPD 2 87f 14.3 Lymphoma 3 74 f 21.9 MI 4 71 m 13.4 pulmonary embolism 5 69 m10.0 lung cancer 6 65 f 10.3 AP control 7 77 f 5.0 Hypertension 8 54 m5.0 Lymphoma 9 81 m 5.5 CHF 10 68 f 6.0 Mammacarcinoma 11 78 f 6.7 MI 1231 m 5.0 Melanoma Age, sex, plasma glucose concentration and diagnosisof type 2 diabetic and control patients.

Leptin decreases β-cell production of IL-1Ra and induces IL-1β releasein human islets. The hypothesis that high concentration of glucose wasresponsible for the in vivo regulation of IL-1Ra expression wasinvestigated in vitro in primary culture of human islets. Exposure ofislets to 33.3 mM glucose for 48 h did neither affect IL-1Ra proteinexpression (FIG. 8A-1 to A-4) nor IL-1Ra release or mRNA expression(FIG. 9A), as compared to controls at 5.5 mM glucose. Because leptinregulates IL-1Ra secretion in monocytes (93), we then postulated that itmay be a regulator of IL-1Ra in β-cells. Exposure of cultured humanislets to 10 nM leptin for 48 hours decreased IL-1Ra expression in mostβ-cells, as determined by double immunostaining (FIGS. 8A-5 & A-6).Western blot analysis of IL-1Ra in human islets revealed that leptindecreased IL-1Ra already after a 15-hour exposure (FIG. 8B).Interestingly, IL-1Ra released into the culture medium and IL-1Ra mRNAwere increased after short-term exposure to 10 to 500 nM leptin for 15hours, but longer exposure for 48 hours resulted in a marked decrease(FIGS. 8C-1 & C-2). A similar dual effect was observed In peripheralblood monocytes that were used as control (FIGS. 8C-3 & C-4).

IL-1Ra action depends on IL-1β, which is why also the regulation ofIL-1β by leptin was studied. Similarly to the regulation of IL-1Ra,exposure of human islets to leptin for 15 hours increased IL-1β releaseinto culture medium (FIG. 8D-1). In contrast to the leptin-induceddecrease of IL-1Ra release after 48 hour, IL-1β release remainedincreased after a 48 hour exposure. Moreover, exposure of peripheralblood monocytes to leptin for 15 hours increased IL-1β release, whereasprolonged exposure for 48 hours did not significantly affect IL-1βrelease (FIG. 8D-2). Quantitative RT-PCR measurements revealed thatIL-1β RNA expression of islets was not significantly changed by leptin.Nevertheless, exposure of cultured human islets to 10 nM leptin for 48hours induced IL-1β expression in TUNEL-positive β-cells, as determinedby triple-immunostaining of islets with anti-IL-1β and anti-insulinantibodies and by the TUNEL assay (FIG. 8E).

Endogenously produced IL-1Ra is a survival factor of β-cells andpreserves β-cell function. We next studied the functional role of IL-1Rain human β-cells by means of siRNA. Two days after exposure of humanislets to siRNA to IL-1Ra (siIL-1Ra), endogenous IL-1Ra RNA expressiondecreased by 69.4±6.5% as compared to islets incubated at 5.5 mM glucosealone, whereas scrambled siRNA had no such effect (FIG. 9A).Antagonization of IL-1Ra was similar at low (5.5 mM) and high (33.3 mM)glucose concentrations (FIG. 9A). SiIL-1Ra dramatically decreased IL-1Raprotein expression inducing an apoptotic process demonstrated by caspase3 activation (FIG. 9B) and β-cell DNA fragmentation (TUNEL-positivenuclei, FIGS. 9C & D). Addition of exogenous rh IL-1Ra prevented theeffect of siIL-1Ra on DNA fragmentation, proving the specificity of theRNA interference (FIGS. 9C & D). Exposure of cultured human islets toelevated glucose concentrations increased the number of β-cellsdisplaying DNA fragmentation (FIG. 9D). This glucose effect was enhancedby co-incubation with siIL-1Ra, whereas exogenous rh IL-1Ra protectedthe β-cells from apoptosis induced by 33.3 mM glucose alone andpartially from 33.3 mM glucose and siIL-1Ra.

Next, the role of endogenous IL-1Ra on β-cell function was evaluated.Antagonizing IL1-Ra by siRNA dramatically decreased the ratio of basalto acute glucose-stimulated insulin release of human islets maintainedat 5.5 mM glucose (FIG. 9E-1). Co-incubation with exogenous rh IL-1Rarestored glucose stimulation. Chronic exposure of human islets to 11.1mM glucose severely blunted acute glucose-stimulated insulin release,which was totally lost in the presence of siIL-1Ra at 11.1 or 33.3 mMglucose and by 33.3 mM alone. Exogenous rh IL-1Ra partially preventedthose effects. Insulin contents of islets exposed to siIL-1Ra and/orhigh glucose concentrations were decreased compared to control (5.5 mMglucose) and partially restored by rh IL-1Ra (FIG. 9E-2)

Leptin induces β-cell apoptosis and impairs β-cell function viaIL-1β-signaling. Exposure of cultured human islets to 10 nM leptin for 4days increased the number of TUNEL-positive β-cells (FIG. 10A).Moreover, leptin enhanced 33.3 mM glucose-induced apoptosis (FIG. 10A).To demonstrate an apoptotic process, in parallel to the TUNEL assay,caspase 3 activation was detected. Exposure of human islets to leptinfor 15 hours did not change baseline cleaved caspase 3 level, but anincrease became apparent after 48 hours (FIG. 10B). Moreover, exposureof leptin for 4 days decreased chronic insulin secretion during theculture period by 41.65% (p<0.01), impaired glucose-stimulated insulinsecretion and decreased insulin content at 5.5 mM glucose, an effectwhich was additive to the deleterious effect of high glucose (FIG. 10C).Finally, to show that leptin-induced DNA fragmentation, impaired β-cellfunction and decreased insulin content are mediated by the dysbalance ofIL-1β and IL-1Ra, human islets were co-incubated with leptin andexogenous rh IL-1Ra. In human islets exposed to leptin, addition of rhIL-1Ra prevented leptin-induced β-cell apoptosis and restored β-cellfunction and insulin content (FIG. 10D).

Chronically elevated blood glucose levels impair the function of β-cellsin the pancreas (3-7;35;78-80). When studying the underlying mechanisms,we have previously observed that exposure of cultured islets to elevatedglucose levels leads to β-cell production and release of IL-1β, followedby impaired β-cell function and death (76). The present data show thatthe IL-1β pathway is not only involved in glucotoxicity but is also amediator of obesity-associated diabetes. Indeed, leptin, the proteinencoded by the ob gene, decreased β-cell expression of IL-1Ra. Inparallel, exposure of human islets to leptin induced IL-1β release.Leptin-induced changes in the IL-1β/IL-1Ra ratio, impaired D-cellfunction and enhanced β-cell apoptosis. The deleterious effects ofleptin can be alleviated by supplementation of exogenous IL-1Ra in asimilar way as it can palliate glucotoxicity.

To the best of our knowledge, this is the first study which showsexpression of IL-1Ra by the β-cells. This is particularly intriguing inthe context of an endocrine organ not primarily belonging to the immunesystem. In addition to the previous findings which show β-cellexpression of IL-1β (76;50), the present observation provides furtherevidence of local inflammatory mediators in the pathophysiology ofdiabetes (94).

Antagonizing endogenous β-cell-IL-1Ra by siRNA led to impaired β-cellfunction and apoptosis. This is not an unspecific effect of siRNA, sincescrambled siRNA was not toxic. It is likely that a certain amount ofIL-1Ra is necessary for the survival of cultured human β-cells.Possibly, IL-1β, which is present in the supernatant of untreatedcultured human islets (76) is ineffective as long as IL-1Ra is expressedand becomes deleterious as soon as the balance changes in its favor. Invivo, it is conceivable that IL-1Ra protects the β-cells from othersources of IL-1i, e. g. the innate immune system (94), and that theβ-cells become particularly vulnerable if leptin decreases itsexpression.

The cellular source of leptin-induced IL-1β of the islet is not clear.Triple immunostaining for IL-1β, insulin and the TUNEL assay of isletsexposed to leptin uncovered only TUNEL-positive β-cells producing IL-1β.Those cells are probably not the unique source of leptin-induced IL-1β.Indeed, rhIL-1Ra blocked leptin-induced apoptosis. Therefore, part ofleptin-induced IL-1β precedes the apoptotic process. Alternatively,islet resident macrophages may well contribute to leptin production,since leptin induces the secretion of IL-1β in peripheral bloodmonocytes, as previously shown (93) and confirmed in the present study.

In normal individuals, the endocrine pancreas responds to an increasedinsulin demand by increasing its function and mass (62;66;95). Thefailure to adapt in diabetes may be partly explained by the deleteriouseffects of chronic exposure to leptin and to elevated glucoseexcursions. Intra-islet production of IL-1β and decreased IL-1Raexpression appear as a final common pathway responsible for impairedβ-cell function and apoptosis. Therefore, therapeutic approachesdesigned to block this pathway may block leptin- and glucotoxicity,preventing a progressive decline in β-cell mass and restoring β-cellfunction. IL-1Ra appears to be a suitable therapeutic agent for thispurpose.

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1. Use of an Interleukin 1 receptor antagonist (IL-1Ra) for thepreparation of a medicament for the treatment or prophylaxis of type 2diabetes in a mammal.
 2. The use according to claim 1, wherein theInterleukin 1 receptor antagonist (IL-1Ra) is a recombinant protein(rIL-1Ra).
 3. The use according to claim 1, wherein the Interleukin 1receptor antagonist (IL-1Ra) is a recombinant human protein (rhIL-1Ra).4. The use according to claim 1, wherein the medicament furthercomprises pyrrolidinedithiocarbamate (PDTC).
 5. The use according toclaim 1, wherein the medicament is adapted for parenteraladministration.
 6. Use of pyrrolidinedithiocarbamate (PDTC) for thepreparation of a medicament for the treatment or prophylaxis of type 2diabetes in a mammal.
 7. The use according to claim 6, wherein themedicament is adapted for parenteral administration.
 8. A method oftreating or prophylactically suppressing type 2 diabetes, the methodcomprising administering to a mammal in need thereof a sufficient amountof an Interleukin 1 receptor antagonist (11-IRa).
 9. The methodaccording to claim 8, wherein the Interleukin 1 receptor antagonist(IL-1Ra) is a recombinant protein (rIL-1Ra).
 10. The method according to9, wherein the Interleukin 1 receptor antagonist (IL-1Ra) is arecombinant human protein (rhIL-1Ra).
 11. The method according to claim8, wherein the medicament further comprises pyrrolidinedithiocarbamate(PDTC).
 12. The method according to claim 8, wherein the medicament isadapted for parenteral administration.
 13. A method of treating orprophylactically suppressing type 2 diabetes, the method comprisingadministering to a mammal in need thereof a sufficient amount ofpyrrolidinedithiocarbamate (PDTC).
 14. The method according to claim 13,wherein the medicament is adapted for parenteral administration.